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IOSR Journal Of Environmental Science, Toxicology And Food Technology (IOSR-JESTFT)
e-ISSN: 2319-2402,p- ISSN: 2319-2399. Volume 5, Issue 2 (Jul. - Aug. 2013), PP 55-58
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Isolation and identification of petroleum degrading bacteria from
oil contaminated soil & water and assessment of their potentiality
in bioremediation
Tazeena Hassan Islam1, Badal Ghosh1, Md. Mehedi Hasan Magnet,
Kaniz Fatema1, Selina Akter1+2, Md.Abdur Rahim Khan1+3,Suvamoy Datta1*
1
Department of Microbiology, Primeasia University, Bangladesh
Department of Microbiology, Jessore Science and Technology University, Bangladesh
3
Essence Homoeopathy Medical Center, 7/A Shobhanbag, Sher-e-Bangla Nagar, Dhaka
2
Abstract : Oil spillage has become a global environmental problem. Natural bioremediation is the only ecofriendly solution to resist its devastating environmental and economic damage damage. In this investigation,
petroleum tolerant and degrading bacteria were isolated from different oil-contaminated soil and water
samples. Bushnell Haas media supplemented with petrol, kerosene, and diesel as sole carbon sources was used
for isolation of bacteria capable of degrading these petroleum fractionates. From three soil samples and two
water samples, a total of nine bacterial strains were isolated capable of degrading petrol, kerosene and diesel
with varying tolerance capacities. The isolates were identified by using standard biochemical tests and
morphological studies, and it was determined that these strains belong to six bacterial genera e.g.,
Staphylococcus, Micrococcus, Streptococcus, Bacillus, Klebsiella and Corynebacterium. The isolated
Staphylococcus spp. were found to be the most tolerant isolate withstanding as high as 7% petroleum. The
others also exhibit tolerance to varying concentrations of petroleum. All these isolates were able to degrade
petroleum completely and produced CO2 within 7 days, with a few exceptions for Bacillus sp. and Klebsiella sp.
which required 15 days for complete degradation of kerosene. These isolates seemed to have potential for
bioremediation of oil contaminated soil and water.
Keywords : Bioremediation, Degradation, Petroleum, Staphylococcus spp., Tolerance
I.
Introduction
Petroleum enters into the environment through waste disposal, accidental spills, leakage tankers and
losses during transport or storage [1]. The extensive use of petroleum products leads to the contamination of
almost all compartments of the environment, and biodegradation of the hydrocarbons by natural populations of
microorganisms has been reported to the main process acting in the depuration of the hydrocarbon-polluted
environments [2]. Annually, around 6 million tons of crude oil from oily compounds enters global
environments, which not only cause human health hazard, but also damage our surrounding environment [3].
The toxic effects of crude oil and refined petroleum oils on plants, animals, humans and the
environment are devastating [4]. Oil toxicity is caused by the soluble fraction, which consists of low boiling
point aromatic hydrocarbon including benzene, toluene, zylene, naphthalene, pentrathene etc. Oil pollution has
been recognized as one of the most serious problems for biotic life. Due to exposure to high oil concentrations,
kidney and liver diseases might develop, cancer is enhanced, and it might also damage bone marrow.
Petroleum hydrocarbon is composed of carbon and hydrogen. Many microorganisms have the ability to
utilize petroleum as sole source of carbon as energy for metabolic activities, and these microorganisms are
widely present and distributed in nature [5]. Bioremediation, which is the removal of organic pollutants from the
environment by microorganisms, is one of the best approaches for restoring contaminants of soil and water.
Bioremediation processes rely on the ability of microorganisms present naturally which are highly
efficient due to their simplicity and cost effectiveness when compared to other technologies. The process of
bioremediation is defined as the use of microorganism to detoxify or remove pollutants, which relies upon
microbial enzymatic activities to transform or degrade offending contaminants. It is an evolving method for the
removal and destruction of many environmental pollutants [6]. Research and application of bioremediation on
soil environment has been started in Indonesia [7]; however in marine environment, it is still need to be studied
due to more complicated aspects and also difficulties. Therefore comprehensive study in biodegradation
processes that involves various factors such as pollutants, microorganisms, energy sources, physical and
chemical conditions, toxicity, competition and metabolites accumulation have to be done. Bioremediation of
petroleum is a mineralization of organic chemicals, leading ultimately to the formation of CO 2, H2O and
biomass. Hydrocarbon utilizing microorganisms are ubiquitously distributed in the environment following oil
spills [8]. Biodegradation has at least three important processes: first, they cause a minor change in an organic
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Isolation and identification of petroleum degrading bacteria from oil contaminated soil & water and
molecule leaving the main structure still impact; second, degradation can also be thought of as fragmentation,
where the original structure of the molecule can still be recognized in the fragments and finally, a complete
mineralization of a compound to inorganic form.
The purpose of this research was to isolate and identify bacteria growing in oil contaminated soils near
petrol and diesel filling pumps of Kamalapur railway station, Buriganga river side and water from Buriganga
river in Dhaka city to degrade petroleum components (petrol, diesel, kerosene). The population of
microorganisms found in oil-polluted environment should degrade petroleum and higher rates than
microorganisms in relatively clean environment [9]. Petroleum hydrocarbon utilizing bacteria should also
intrinsically tolerate oil contaminated environment because of their extended exposure and the capability to
utilize oil as energy source.
II.
Materials and Methods
2.1. Sample Collection
Three soil samples and two water samples were collected from different oil contaminated sites in sterile
bottles and polythene bags. Samples were collected in 5 cm depth from the surface of soil to avoid surface
contamination. Water samples were collected in oil contamination site. Collected samples were transferred into
the laboratory under sterile condition and stored at 4˚C until processed for analysis.
2.2. Isolation of bacterial cultures
Ten gm of soil and 10 ml water samples were diluted into 90 ml distilled water and prepared dilutions
(10ˉ3, 10ˉ4, 10ˉ5). Then 0.1 ml suspension from each dilution was transferred into nutrient agar plate by using
spread plate technique and incubated at 37˚C for 24 h.
2.3. Isolation and identification of petroleum degrading bacteria
Bushnell Haas (BH) agar/broth medium was used for the isolation of petroleum degrading bacteria.
This medium contains all nutrients except the carbon source. The medium composition is as follows: MgSO4
.7H2O (0.2 g/L), K2HPO4 (1.0 g/L), KH2PO4 (1.0 g/L), FeCl3 (0.05 g/L), NH4NO3 (1.0 g/L) and CaCl2 (0.02
g/L) with pH 7.2. Agar (20 g/L) was added for BH agar [10, 11]. The medium was supplemented with
petroleum to serve as the only source of carbon and energy. Isolated bacterial cultures were streaked on BH agar
plate and 100 µl of petroleum was spread onto the surface of agar plate as sole source of carbon. The plates were
incubated at 37˚c for 2-3 weeks. After the incubation period, only petroleum degrading bacteria remained on the
surface of the plates [12].
The bacterial colonies were then identified by Gram staining and biochemical tests according to
Bergey´s manual. For more confirmation of petroleum degradation, BH broth medium was used. Isolated
bacterial cultures were enriched into peptone broth. 1 ml of enriched culture was added into the test tube
containing BH broth, 50 µl of petroleum was added as sole source of carbon in each of the tubes. All the test
tubes were incubated at 37˚C for 2-3 weeks. Petroleum degrading bacteria will produce gas bubbles on the
petroleum-water interface, which indicate a positive degradation reaction.
2.4. Tolerance of Petroleum Concentrations
Tolerance to petroleum (diesel, petrol, kerosene) concentrations were assessed by the ability of
organisms to grow on BH agar plate into which petroleum (0.1- 7.0% concentrations) were incorporated.
Different concentrations of petroleum containing BH agar plates were inoculated with the isolated cultures. The
inoculated agar plates were incubated at 37˚C for 7 days. After the incubation period, tolerance range of
petroleum concentrations of isolated bacteria were obtained on the basis of their growth.
2.6. Identification of isolates
Morphological and biochemical tests were done for identification of isolates
III.
Results and Discussion
In this research, we used soil and water samples from different oil contaminated sites for isolation and
identification of petroleum degrading bacteria. Isolated bacteria were able to utilize the three petroleum
components i.e. petrol, diesel and kerosene tested as their sole carbon source. A total of nine petroleum
degrading bacteria were isolated and were identified by standard biochemical tests and morphological studies
(Table 1).
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Isolation and identification of petroleum degrading bacteria from oil contaminated soil & water and
Isolate
designation
Gram
stain
Shape
Gluco
se
Lacto
se
H2S test
Ferm
entati
on
Indole test
MR test
VP test
Citrate test
Urease test
Catalase test
Table 1: Gram reaction and biochemical characterization of petroleum degrading bacterial isolates.
Psw
Psy
Rsw
Rsy
Ksw
Ksy
Saw
Say
Suw
Suy
G(+)
G(+)
G(+)
G(+)
G(+)
G(+)
G(+)
G(-)
G(+)
G(-)
Cocci
Cocci
Cocci
Cocci
Cocci
Cocci
Rod
Rod
Rod
Rod
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
-
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
-
Presumptive Organisms
Streptococcus spp.
Micrococcus spp.
Micrococcus spp.
Staphylococcus spp.
Staphylococcus spp.
Staphylococcus spp.
Bacillus spp.
Klebsiella spp.
Corynebacterium spp.
Escherichia spp.
On the basis of these studies, it was determined that these strains belong to six bacterial genera e.g.,
Staphylococcus, Micrococcus, Streptococcus, Bacillus, Klebsiella and Corynebacterium. All these isolates were
found to grow effectively on BH agar supplemented with different petroleum fractionates (Fig. 1). In BH broth,
those isolates were found to degrade petroleum with CO2 production within 7 days, except for Bacillus sp. and
Klebsiella sp. which required 15 days for degradation of kerosene (Fig. 2).
Figure 1: Graphical representation for the growth of different isolates on BH agar with
different petroleum fractionates as sole carbon source. The X-axis designates the days of
incubation and in the Y-axis designates test isolates.
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Isolation and identification of petroleum degrading bacteria from oil contaminated soil & water and
Figure 2: Graphical representation for the degradation of different isolates on BH broth with a layer of
different petroleum fractionates as sole carbon source. The Y-axis designates the days of incubation. The
positive degradation was observed by the accumulation of gas (CO2) bubbles at the media-petroleum interface.
Petroleum degrading bacteria also shows tolerance to considerable concentrations of petroleum, which
is a vital criterion to act in bioremediation of highly polluted environments. All the test isolates were also found
to tolerate petrol, diesel and kerosene in different concentrations ranged between 0.1 and 7% (Fig. 3). The
Staphylococcus spp. was found to be the most tolerant withstanding as high as 7% of petroleum. All these
isolates could be potentially used for bioremediation purposes for cleaning up oil polluted area.
Figure 3: Graphical representation for the growth of different isolates on BH agar with different
petroleum fractionates as sole carbon source. The X-axis designates the days of incubation and in the
Y-axis designates test isolates.
IV.
Conclusion
In this study, three petroleum degrading bacterial genera e.g., Micrococcus, Staphylococcus,
Streptococcus were isolated from soil. The other three genera e.g., Bacillus, Klebsiella and Corynebacterium
were isolated from water. A Staphylococcus sp. isolated from Kamalapur soil was found to tolerate the highest
concentrations (7%) of petroleum, therefore can be used potentially to decontaminate oil spillage from water
and soil. Both Gram positive and negative bacterial genera were isolated and assumed to have a common pattern
of tolerance and degradation of petroleum.
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